Endogenous cannabinoid (eCB) signaling in the brain plays a homeostatic role in the constraint and termination of stress responses. During the previous cycle of the TSRI-ARC we found that chronic intermittent EtOH exposure down-regulates eCB signaling in the central nucleus of the amygdala, a brain region critically involved in stress responses and emotional processing. We also found that dependenceassociated anxiety-like behavior and excessive EtOH consumption are alleviated by enhancement of eCB tone. Endocannabinoids are also present in other stress-responsive brain regions such as the basolateral amygdala (BLA) and bed nucleus of the stria terminalis (BNST) where they play a prominent role in the plasticity of excitatory and inhibitory signaling. Dysregulated synaptic function in these regions is believed to contribute to dependence-associated affective disorders and we have gathered preliminary evidence that eCB clearance mechanisms are disrupted in these regions by alcohol dependence. Based on these observations we hypothesize that dysregulated eCB signaling in the BLA and BNST contributes to affective dysregulation and excessive EtOH consumption associated with long-term EtOH exposure. This hypothesis will be tested through three Specific Aims. Aim 1 will employ biochemical and neurochemical approaches to characterize the nature and persistence of dysregulated eCB function resulting from excessive EtOH exposure. Aim 2 will characterize the influence of disrupted eCB function in the BLA and BNST on dependence-associated anxiety-like behavior over a period of protracted abstinence. The relative influence of two primary eCB molecules, 2-AG and anandamide (AEA) will be characterized using pharmacological and genetic manipulations of their respective clearance mechanisms. Aim 3 will characterize the efficacy of selective eCB clearance inhibitors for reducing high levels of EtOH consumption associated with dependence and protracted withdrawal. The experimental design incorporates two distinct animal models of excessive drinking to index the development of eCB disruptions along the trajectory from chronic binge drinking to excessive EtOH consumption motivated by dependence.